| The term 'retinitis pigmentosa (RP)' refers to a group of genetically mediate,degenerative retinal diseases, that display the same pathological changes of progressively dysfunction of photoreceptors and RPEs,and characterized with night blindness,constriction of vision field and finally the loss of central vision. Despite the fact that over 60 mutations have been discovered so far, the biological mechanism involved has not yet been utterly understood. Fortunately, label-free quantification-based proteomics analysis therewith its advantage of more economic and simpler procedures have been increasingly adopted in modern biological researches. With differential proteomics, changes in abundance of proteins in disease state and health state at different stage of retinitis pigmentosa can be identified.These proteins could be used in studying the significant factors involved in disease.In this study, we implied MS-based label-free quantification proteomics approaches to identify the changes in abundance of proteins in dystrophic RCS rats, compared with non-dystrophic congenic controls, at four age stage (postnatal day 18/24/30/36), based on Linear Trap Quadrupole (LTQ) Orbitrap Velos Pro MS combined with Liquid Chromatography (LC). Bioinformatics approaches were then performed on these identified differentially expressed proteins (DEPs) to dig into the potential protein-protein interaction and biological molecular pathways involved in retinal degeneration.In the first chapter, we introduce research background and actuality of retinitis pigmentosa, proteomics and differential proteomics, highlight research significance, content and technique of differential proteomics applied to retinitis pigmentosa.In the second chapter, we illustrate the work of protein identification of dystrophic RCS rats and non-dystrophic congenic controls through label-free quantification, followed by differential proteins filtering with independent-samples students t-test. After retinal protein extraction, SDS-PAGE, in-gel digestion with trypsin, the sample's were analyzed by LC-MS/MS. Each experimental group contains two pooled protein lysates from sets of 4 retinas obtained from 2 individual. To increase the peptide coverage and experimental reliability, we carried out four independent experiments on each retinal protein sample. The raw data obtained from LC-MS/MS is searched in Rattus norvegicus fasta database from Uniprot and analyzed by MaxQuant software and MASCOT software. The result indicates that proteome dataset from MaxQuant software have a greater repetitive rate and reliability than that from MASCOT software. Then we performed independent-samples t-test on proteome datasets outputted by MaxQuant software and 434 DEPs between dystrophic RCS rat and non-dystrophic congenic controls have been identified.The third chapter in this study mainly focuses on bioinformatics analysis on DEPs in RCS rat retina. Enriched analysis of Gene Ontology annotation indicated that most of differential proteins were localized on cytosol and mitochondria, and participated in cellular metabolism process, with molecular function of protein binding and catalytic activity.Enriched analysis of KEGG pathway annotation indicated that glycolysis pathway shows significance different. Finally, upstream regulatory analysis indicated that the transcription factor E2F1 may plays an important role in regulating most of the DEPs.Combined with the results of differential proteomics and bioinformatics analysis on RCS rat retina, we identified mitochondria and glycolysis pathway as the key initiation factors in early retinal degenerative process and the transcription factor E2F1 made a substantial contribution to the disease etiology. Thus,it provide a new prospective to the therapeutic approaches for this retinal degenerative disease. |
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